<2021-12-07 Tue> David biweekly meeting

projects

successfully correlated actin-positive beads between fluorescence and EM
- light microscope field (bright field, Sac6-GFP, far-red lipid)
- bead + actin at top right:
- bead with very low actin signal at middle left:
sadly, we could not make out lipid bilayers in these TEM micrographs, so can't make any interpretations that we care about
- a clear bilayer vesicle in actin network next to a bead with ambiguous SLB:
- zoomed in on very low actin bead with no discernable lipid bilayer:
  
  $2021-12-07_11-48-52_Screen Shot 2021-12-07 at 11.32.27 AM.png$
- lots of sections from this sample were laid on top of grid bars and therefore inaccessible, so perhaps we got unlucky with only having sections at a glancing angle with the SLB
- they will use a different type of grid next time to avoid blocking important regions, but that comes with the risk of losing the entire sample if a region gets irradiated too hard
currently communicating with tech support to fix Wall-E laser, not sure how long this will take

fix wall-E and image negative control beads for correlation
finish sectioning and image GFP-positive beads
determine whether there is a difference in membrane shape between high and low fluorescence beads
- come up with some metric of scoring if so
if there are differences, collect triplicate data and do statistical test

we will pick this direction back up for Kahmina's rotation, focusing on the early CME protein module as inspired by &day_liquid-like_2019
requested Eps15 and Fcho1 strains from Stachowiak lab to serve as positive controls, but also started cloning our own constructs to go with whatever comes through first

clone Eps15 expression construct in E. coli
purify Eps15 according to published procedure
fluorescently label Eps15
perform positive control in vitro Eps15 droplet experiments
1. time lapses for fusion and relaxing
2. FRAP
3. tether to SLBs or GUVs
express/purify Ede1 in S. cerevisiae and E. coli in parallel to see which approach is more amenable to purification
repeat in vitro droplet experiments with Ede1
reconstitute Ede1 cytoplasmic condensates in cytoplasmic extracts of Ede1-overexpressing strain
try to fuse different yeast strains with Ede1 condensates tagged with different fluorescent proteins

fixed mistakes from energies in minimal model, so it's generally relaxing how I would expect it to
- the first and last points are immobile, everything else is allowed to move
- there is turgor pressure, membrane tension, and bending rigidity
the fact that the membrane bows outward from turgor tells us that we need to have both yeast-like and mammalian-like models or pick one
- we would need to add a cell wall if we keep turgor pressure
- we would need to remove turgor pressure if there's no cell wall
I want to think carefully about which biological system is more relevant to this mechanical influence of the droplet hypothesis
- I'm worried that the forces required for yeast endocytosis is so high that it's really unlikely for the force from a droplet to be relevant
- maybe this is a good discussion to have with my thesis committee